Toxoplasma gondii is an obligate intracellular protozoan parasite which is the leading cause of focal central nervous system infections in patients with AIDS and Causes devastating congenital infections. The parasite resides within cells inside a specialized membrane bound vacuole which does not fuse with host organelles of the endocytic cascade but has tightly opposed mitochondria and endoplasmic reticulum from the host cell. The vacuolar space and parasitophorous vacuole membrane surrounding the intracellular parasite are modified by protein secretion from parasite dense granules. It is our hypothesis that parasite viability and replication are absolutely dependent upon both organelle association and dense granule secretion, and that intervening in these processes will block parasite growth. To address the mechanism and consequences of organelle association, interacting proteins in the parasitophorous vacuole and organelle membranes which mediate the association will be identified and characterized. The physiological relevance of the association will be explored by attempting to disrupt the association. To address the process of protein delivery into and secretion from dense granules, the signals which mediate or exclude delivery of proteins into dense granules will investigated, and the machinery which controls dense granule secretion will be identified. Manipulations which block delivery of secretion of dense granule proteins will be tested for their effects on parasite viability. These experiments explore two processes which are certain to be critical for successful infection with T. Gondii, and will provide insight into these novel mechanisms for intracellular parasitism.